Roles Of EphB1 In Lung Cancer Cell Dormancy/reactivation And Tumor Immunity

Objectives:At present,platinum-based chemotherapy has been the first-line treatment for advanced non-small cell lung cancer patients.The tolerance of lung cancer cells to chemotherapy is the main cause of therapeutic failure.Studies have found that the dormancy of lung cancer cells is closely related to the resistance of lung cancer cells to chemotherapy.The dormant and reactivated cancer cells are the key sources of tumor progress and metastasis recurrence after chemotherapy.As a member of the receptor tyrosine family,Eph B1 can not only participate in cell growth,differentiation,embryonic development and intracellular signal transmission,but also play an important role in lung cancer development and cancer immune.The phosphorylated signal of Ephrin B ligands-Eph B1 receptor between adjacent cells plays an important role in these pathological processes.The roles of cis signal transduction initiated by the oligomerization of ligand-independent Eph receptors has not been fully studied,which may represent an important regulatory mechanism.In current study,we have explored the specific biological characteristics of dormant and reactivated cancer cells induced by cisplatin treatment.We have also examined the roles of Eph B1 in chemotherapeutic drugs-induced lung cancer cell dormancy and reactivation and its regulatory mechanism.In this study,we used Eph B1knockout mice to study the influence and regulation mechanism of EphB1 on the tumor immune microenvironment and lung cancer development.Intervention in Eph B1 cis and trans signals is expected to become effective therapeutic strategies in cancer therapy.The lead compound targeting Eph B1 kinase domain or the JMS region was obtained by the drug virtual screening technology,and their roles were examined in the tumor dormancy/reactivation and tumor immunity.Method:In this study,we introduced a cell model that simulates the cisplatin response process of patients with non-small cell lung cancer（NSCLC）.Cell cycle,EMT-MET and dormancy-reactivation-related molecules at protein and RNA levels were measured.The dormant and reactivated lung cancer cells were subjected to RNA-seq and ATAC-seq to analyze the effect of cisplatin-induced dormancy and reactivation on the gene expression profiles and the regulatory effect of chromatin accessibility.RT-PCR and Western Blot were used to detect the expression of Eph B1 and p-Eph B1.After discovering the difference in dynamic expression of Eph B1 and p-Eph B1,the mutants of Eph B1 were constructed and the roles of the cis and trans signals of Eph B1 were studied.We constructed Eph B1 knockout mice and used flow cytometry to detect the proportion of immune cells in these unprocessed tissues.In the LLC subcutaneous tumor model,the flow cytometry was used to detect the proportion of immune cells of the peripheral blood,bone marrow and spleen tissues in the mice.Ephrin B2-Fc,which increases Eph B1 trans signal,was used to treat the bone marrow cells of wild-type mice in vitro to detect DC cell differentiation.We selected CD11C positive cells for RNA-seq and conducted relevant analysis and pathway verification.We verified three Eph B1 tyrosine kinase inhibitors obtained from literature through CCK8 experiment to examine its growth effect on cisplatin’s dormant/recovery lung cancer cells;The expression of SOX2,NANOG in the lung cancer cells and the expression of DUSP1 of MAPK pathway in the mice bone marrow were examined by western blot analysis;Flow Cytometry was used to detect the effects of three kinds of kinase antagonists on DC.We have applied virtual screening technology to obtain lead compounds Polymyxin B and Leuprorelin that target Eph B1 JMS region;The effect of these lead compounds on the growth of dormant/reactivated tumor cells induced by cisplatin was examined by CCK8 assay;The regulatory roles of Polymyxin B that target Eph B1 JMS region were examined by Western blot.Results:We found that during the dormant and reactivated process of lung cancer cells induced by cisplatin,the cells showed EMT-MET phenotypes.The Real-Time PCR and Western Blot assay showed that EMT-MET related molecules,dormant-reactivated related molecules and stem cell related molecules expressed in dynamic expression.RNA-seq analysis revealed that the dynamic regulatory network dominated the process of enrichment of cancer stem cells.Among them,NANOG was up-regulated in the dormant tumor cells and SOX2 was up-regulated in the reactivated tumor cells.The combined analysis of ATAC-seq sequence and motif showed that OCT4-SOX2-TCF-NANOG motif was related to the enrichment of cisplatin-induced lung cancer stem cells.In addition,we found that Eph B1 and p-Eph B1 showed dynamic expression patterns during lung cancer cell dormancy and reactivation.Tumor cells with less cell-cell contacted exhibited dormant and EMT states,in which ligand-independent Eph B1 promoted lung cancer cells to enter dormant state by activating p-p38 and down-regulating E-cadherin.On the contrary,in the state of MET,when the adhesion between cells was restored,Eph B1 and ligand Ephrin B2 up-regulated the expression of NANOG and SOX2 through trans signals which promoted the stemness of tumor cells.We used Eph B1 mutants to elucidate the key domains of Eph B1 cis and trans regulation.The flow cytometry analysis of WT and EphB1^-/-mice showed that compared with the WT group mice,the Eph B1^-/-mice had no obvious differences in immune cells such as dendritic cells and macrophages in these tissues.In the subcutaneous transplanted tumor model,the tumor formation rate and growth rate of LLC cells in Eph B1^-/-mice were significantly higher than those in wild-type mice.The flow cytometry showed that the proportion of macrophages and DC decreased;the proportion of M-MDSC increased in the Eph B1^-/-tumor model.Ephrin B2-Fc was found to increase the proportion of c DCs groups.We found that the MAPK signaling pathway was enriched in Eph B1^-/-DC by RNA-seq.Ephrin B2-FC treatment of mice bone marrow cells can promote DUSP1 expression and decrease the expression of p-p38.CCK-8 assay revealed that chlotetracycline displayed specific growth inhibitory roles in dormant and reactivated tumor cells;Eph B1 tyrosine kinase inhibitors can significantly inhibit the expression of P-Eph B1 and SOX2 of lung cancer cells;Western blot results show that Eph B1 tyrosine kinase inhibitors can inhibit the expression of DUSP1 in the MAPK signaling pathway in BMDC.The small molecules targeting regions near JMS-polyphonin B and Leuprorelin were obtained from virtual screening.High-concentration of Leuprorelin can inhibit the growth of reactivated tumor cells,while the low-concentration of Polymyxin B inhibited the growth of the dormant tumor cells induced by cisplatin;Western blot assays showed that Polymyxin B can specifically inhibit the expression of p-Eph B1 and SOX2 during dormancy.Conclusion:1.Cisplatin can induce lung cancer cells into the states of dormance-reactivation,EMT-MET,and induce the cancer stem cell enrichment;2.Tumor cells with less cell-cell contact exhibit dormant and EMT states,in which ligand-independent Eph B1 promotes lung cancer cells to enter dormant state by activating p-p38 and down-regulating E-cadherin;In the reactivated state,when the cell-cell contacts re-established,the Eph B1 trans signal promotes the expression of SOX2and Nanog and the enrichment of cancer stem cells;3.The domains of LBD,CRD and FNIII are important to the oligomerization of Eph receptor and following cis signals activation.4.Eph B1 trans affects the maturity and differentiation of DC through the p38 MAPK signaling pathway.5.The inhibitors of targeting Eph B1 tyrosine kinase domain can significantly inhibit the expression of p-Eph B1 and SOX2;Among them,Chlotetracycline can significantly inhibit the proliferation of the dormant/reactivated tumor cell induced by cisplatin and Demecycline can obviously inhibit the differentiation and maturity of DC;6.The lead compound compound targeting Eph B1 JMS neighboring area polymyxin B obviously inhibits the proliferation of cisplatin-induced dormant cells;Leuprorelin specifically inhibits the proliferation of reactivated cells induced by cisplatin.7.The lead compound compounds targeting Eph B1JMS neighboring area are better than those targeting Eph B1 tyrosine kinase domain for the targeted inhibitory effect.